Compositions and methods for the identification of inhibitors of protein synthesis

ABSTRACT

Compositions and methods for identifying inhibitors of RNA-target molecule interactions are provided as well as identifying inhibitors that block the role of tRNA in protein synthesis. The methods involve forming a mixture comprising a tRNA fragment molecule containing a modified nucleotide, a target molecule capable of binding to the tRNA fragment, and a test compound. The mixture is incubated under conditions that allow binding of the tRNA and the target molecule in the absence of the test compound. Assays can then be performed that detect whether or not the test compound inhibits the binding of the tRNA molecule and the target molecule. High throughput assays are also provided.

CROSS-REFERENCE TO RELATED APPLICATONS

This application is a divisional application of U.S. patent applicatonSer. No. 11/943,924 filed on Nov. 21, 2007 and subsequently issued asU.S. Pat. No. 7,598,040 on Oct. 6, 2009, which further claims thepriority of U.S. Provisional Application No. 60/866,988, filed Nov. 22,2006 and U.S. Provisional Application No. 60/867,263, filed Nov. 27,2006, all of which are incorporated herein by reference in theirrespective entireties.

FIELD

The invention generally relates to compositions and methods ofidentification of inhibitors of protein synthesis.

BACKGROUND

The significant progress in the understanding of the molecular basis ofhuman disease in the last few decades has led to a significant increasein the number of potential therapeutic targets. Over the years manydrugs have been developed that target various biological processes suchas enzymatic reactions and signal receptors. Among these, proteinsynthesis inhibitors represent a large potential class of moleculartargets with importance that spans across many therapeutic areas, suchas antibiotics, antivirals, and anticancer treatments.

One class of under utilized therapeutic targets is the ribonucleic acids(RNAs) involved in bacterial protein synthesis. More specifically, theprotein synthesis processes that use post-transcriptional modified RNAnucleotides as substrates may be ideal therapeutic targets.Post-transcriptional nucleotide modifications can be as simple as theaddition of a methyl group to a standard nucleotide or complexmulti-step addition of amino acid like side chains (Soli, D. andRajBhandary, U. L., tRNA: Structure, Biosynthesis, and Function, ASMPress 1995; Grosjean, H. and Benne, R., Modification and Editing ofRNA., Washington, D.C., ASM Press, 1998). While 1 to 2% of all RNA basesare modified, the nucleotides in the active sites of the ribosome andthe transfer RNA (tRNA) that interact with the ribosome are modified at10-fold the rate of modification outside these active sites. One of thefunctions of these modifications is to enhance the selectivity andspecificity of the RNA:enzyme interactions that occur duringtranscription and translation. One such class of enzymes that utilizetRNA containing modified nucleotide bases is the amino acyl tRNAsynthetases (AaRS) which catalyze the attachment of a specific aminoacid to its corresponding tRNA. The specificity of the AaRS to theirrespective amino acid can be influenced by many features of the tRNAmolecule including the modified nucleotide bases. In the case of theLysine tRNA synthetase (LysRS), the anticodon stem loop (ASL) of thetRNA contains 2 or 3 modified nucleotide bases depending upon theorganism.

There is a need for the identification of inhibitors of proteinsynthesis from RNA molecules having modified nucleotide bases. To thisend, there also remains a need for the development of methods for theidentification of such inhibitors. Such inhibitors may be useful for thedevelopment of, for example, antimicrobial compounds for use intherapeutic applications.

SUMMARY

Compositions and methods are provided for the identification ofinhibitors of protein synthesis. In one aspect, methods of identifyingan inhibitor of tRNA-target molecule binding, comprise the steps of (1)forming a mixture of a first nucleic acid molecule comprising a tRNATΨC-loop fragment, a target molecule capable of binding to the tRNATΨC-loop fragment, and a test compound, where the tRNA TΨC-loop fragmentcontains at least one modified nucleotide; (2) incubating the mixtureunder conditions that allow binding of the tRNA TΨC-loop fragment andthe target molecule in the absence of the test compound; and (3)detecting whether or not the test compound inhibits the binding of thetRNA TΨC-loop fragment and the target molecule, wherein the absence ofbinding of the tRNA fragment and the target molecule is indicative ofthe test compound being an inhibitor of tRNA-target molecule binding.

In another aspect, methods of identifying an inhibitor of tRNA-targetmolecule interaction, comprise the steps of (1) forming a mixturecomprising a first nucleic acid molecule comprising a tRNA D-loopfragment, a target molecule capable of binding to the tRNA D-loopfragment, and a test compound, where the tRNA D-loop fragment containsat least one modified nucleotide; (2) incubating the mixture underconditions that allow binding of the tRNA D-loop fragment and the targetmolecule in the absence of the test compound; and (3) detecting whetheror not the test compound inhibits the binding of the tRNA D-loopfragment and the target molecule, where the absence of binding of thetRNA fragment and the target molecule is indicative of the test compoundbeing an inhibitor of tRNA-target molecule interaction.

In a further aspect, methods of diagnosing whether a subject has abacterial infection, comprise the steps of (1) forming a mixturecomprising a first nucleic acid molecule derived from a fragment of atRNA molecule comprising at least one modified nucleotide and abiological sample from a subject, where the fragment tRNA moleculecorresponds to a TΨC-loop, a D-loop, or an anticodon stem loopstructure; (2) incubating the mixture under conditions that allowbinding of the first nucleic acid molecule and a target moleculeindicative of the presence of a bacteria in the absence of the testcompound; and (3) detecting whether the first nucleic acid moleculebinds to the target molecule, where the binding of the first nucleicacid molecule and the target molecule is indicative of the positivediagnosis of a bacterial infection in the biological sample.

In another aspect, microarrays are provided comprising a plurality ofnucleic acid molecules having at least one modified nucleotide and asolid support to which the plurality of nucleic acid molecules areattached.

In another aspect, isolated nucleic acid molecules are providedcomprising a nucleic acid fragment derived from a tRNA D-loop having atleast one modified nucleotide. In still another aspect, isolated nucleicacid molecules are provided comprising a nucleic acid fragment derivedfrom a tRNA TΨC-loop having at least one modified nucleotide.

In a further aspect, methods of identifying a target molecule that bindsto a RNA molecule are provided comprising the steps of (1) forming amixture comprising a biological sample and at least one nucleic acidmolecule derived from or corresponding to a tRNA loop having at leastone modified nucleotide, where the tRNA loop is selected from the groupconsisting of a TΨC-loop, a D-loop, and an anticodon loop; (2)incubating the mixture under conditions that allow binding of a targetmolecule in the biological sample to the first nucleic acid molecule;and (3) detecting whether a target molecule binds to the first nucleicacid molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-D provide an amino acid sequence alignment of the LysRSsequences from food and water borne bacteria species included on theCategory B pathogen list compared to human sequence. The LysRS sequenceis highly conserved across 3 of the 7 species and has very littlehomology with the human sequence.

FIG. 2 provides the sequences of RNA oligomers containing variousmodified nucleotides used in binding experiments using a peptide mimicof phenylalanine synthetase, as well as the results of bindingexperiments using the phenylalanine synthetase peptide mimic.

FIG. 3 provides representative sequences for tRNA fragments containingvarious modified nucleotide bases. Motif 1 represents one embodiment ofa tRNA anticodon-stem loop structure containing modified nucleotides.Motif 2 represents one embodiment of a tRNA TΨC loop fragment containingmodified nucleotides. Motif 3 represents one embodiment of a tRNA D-loopfragment containing modified nucleotides. Motif 4 represents oneembodiment of a linear tRNA-anticodon fragment containing modifiednucleotides.

FIG. 4 provides, in one aspect, a schematic of a process for developingan array having nucleic acid molecules containing modified nucleotidesand interrogating the array with target molecules.

DETAILED DESCRIPTION

The present disclosure relates to compositions and methods for theidentification of compounds useful for inhibiting protein synthesis. Thecompositions and methods are useful for the identification of inhibitorsof the interactions of RNA having one or more modified nucleotide baseswith a target molecule. Thus, the disclosure generally relates tocompositions and methods for the identification of inhibitors ofRNA-target molecule interactions.

Prior to describing this invention in further detail, however, thefollowing terms will first be defined.

DEFINITIONS

As used herein, an “inhibitor” refers to any compound capable ofpreventing, reducing, or restricting the interaction or binding of anucleic acid molecule having at least one modified nucleotide base to atarget molecule. An inhibitor may inhibit such interaction or binding,for example, by preventing, reducing or restricting binding of nucleicacid molecule to the binding site of a target molecule. In someembodiments, the inhibition is at least 20% (e.g., at least 50%, 70%,80%, 90%, 95%, 98%, 99%, 99.5%) of the binding as compared to thebinding in the absence of the inhibitor. In one aspect, an inhibitorprevents, reduces, or restricts the binding of a transfer RNA (tRNA), orfragment thereof, to a target molecule. Assays for analyzing inhibitionare described herein.

As used herein, a “label” or “detectable label” is any composition thatis detectable, either directly or indirectly for example, byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans). Useful labels include, but are not limited to, radioactiveisotopes (for example, ³²P, ³⁵S, and ³H), dyes, fluorescent dyes (forexample, Cy5 and Cy3), fluorophores (for example, fluorescein),electron-dense reagents, enzymes and their substrates (for example, ascommonly used in enzyme-linked immunoassays, such as, alkalinephosphatase and horse radish peroxidase), biotin-streptavidin,digoxigenin, or hapten; and proteins for which antisera or monoclonalantibodies are available. Moreover, a label or detectable moiety caninclude an “affinity tag” that, when coupled with the target moleculeand incubated with a test compound or compound library, allows for theaffinity capture of the target molecule along with molecules bound tothe target molecule. One skilled in the art will appreciate that anaffinity tag bound to the target molecule has, by definition, acomplimentary ligand coupled to a solid support that allows for itscapture. For example, useful affinity tags and complimentary partnersinclude, but are not limited to, biotin-streptavidin, complimentarynucleic acid fragments (for example, oligo dT-oligo dA, oligo T-oligo A,oligo dG-oligo dC, oligo G-oligo C), aptamers, or haptens and proteinsfor which antisera or monoclonal antibodies are available. The label ordetectable moiety is typically bound, either covalently, through alinker or chemical bound, or through ionic, van der Waals or hydrogenbonds to the molecule to be detected.

As used herein, a “modified nucleotide” refers to any modification of anucleotide base. Modified nucleotide bases include, but are not limitedto, but not limited to, unknown modified adenosine (?A);1-methyladenosine (m1A); 2-methyladenosine (m2A);N⁶-isopentenyladenosine (i6A); 2-methylthio-N⁶-isopentenyladenosine(ms2i6A); N⁶-methyladenosine (m6A); N⁶-threonylcarbamoyladenosine (t6A);N⁶-methyl-N⁶-threonylcarbomoyladenosine (m6t6A);2-methylthio-N⁶-threonylcarbamoyladenosine (ms2t6A);2′-O-methyladenosine (Am); I Inosine (I); 1-methylinosine Ar(m1I);2′-O-(5-phospho)ribosyladenosine (Ar(p));N⁶-(cis-hydroxyisopentenyl)adenosine (io6A); Unknown modified cytidine(?C); 2-thiocytidine (s2C); 2′-O-methylcytidine (Cm); N⁴-acetylcytidine(ac4C); 5-methylcytidine (m5C); 3-methylcytidine (m3C); lysidine (k2C);5-formylcytidin (f5C); 2′-O-methyl-5-formylcytidin (f5 Cm); unknownmodified guanosine (?G); 2′-O-(5-phospho)ribosylguanosine (Gr(p));1-methylguanosine (m1G); N²-methylguanosine (m2G); 2′-O-methylguanosine(Gm); N²N²-dimethylguanosine (m22G); N²,N²,2′-O-trimethylguanosine(m22Gm); 7-methylguanosine (m7G); archaeosine (fa7d7G); queuosine (Q);mannosyl-queuosine (manQ); galactosyl-queuosine (galQ); wybutosine (yW);peroxywybutosine (o2yW); unknown modified uridine (?U);5-methylaminomethyluridine (mnm5U); 2-thiouridine (s2U);2′-O-methyluridine (Um); 4-thiouridine (s4U); 5-carbamoylmethyluridine(ncm5U); 5-methoxycarbonylmethyluridine (mcm5U);5-methylaminomethyl-2-thiouridine (mnm5s2U);5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U); uridine 5-oxyaceticacid (cmo5U); 5-methoxyuridine (mo5U); 5-carboxymethylaminomethyluridine(cmnm5U); 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U);3-(3-amino-3-carboxypropyl)uridine (acp3U);5-(carboxyhydroxymethyl)uridinemethyl ester (mchm5U);5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um);5-carbamoylmethyl-2′-O-methyluridine (ncm5Um); Dihydrouridine (D);pseudouridine (Ψ); 1-methylpseudouridine (m1Ψ); 2′-O-methylpseudouridine(Ψm); ribosylthymine (m5U); 5-methyl-2-thiouridine (m5s2U); and5,2′-O-dimethyluridine (m5Um).

As used herein, a “target molecule” refers to any biological molecule ofinterest or molecule that is capable of binding to a nucleic acidmolecule having at least one modified nucleotide under physiologicalconditions. The biological molecule of interest can be any class ofbiological molecule, including, but not limited to, ligands, receptors,peptides, proteins, enzymes, polypeptides, nucleic acids(oligonucleotide or polynucleotide of RNA or DNA), antibodies, epitopes,hormones, oligosaccharides, or any other biological molecules. In oneaspect, target protein molecules include proteins involved in proteinsynthesis, including proteins involved in RNA maturation and proteinsinvolved in translation. Particular target molecules include anymolecule involved in protein synthesis, such as, for example, aminoacyltRNA synthetases, methyl transferases, pseudouridine synthases,ribosomes, retroviral reverse transcriptases, messenger RNAs, tRNA,viral RNA or fragments thereof. For example, if transcripts of genes arethe interest of an experiment, the target molecules would include mRNA.Other examples include protein fragments, small molecules, etc. “Targetnucleic acid” refers to a nucleic acid (often derived from a biologicalsample) of interest that is capable of binding, or otherwise interactingwith, an RNA molecule. Target nucleic acid molecules include RNA and DNAmolecules involved in binding to tRNA loop sequences having at least onemodified nucleotide. A target molecule may be detected using one or morenucleic acid molecules having at least one modified nucleotide.

Methods are provided for identifying inhibitors of RNA binding to atarget molecule. The methods generally involve forming a mixture of aRNA molecule having at least one modified nucleotide, a target moleculecapable of binding to the RNA molecule and a test compound, andincubating the mixture under conditions that allow the binding of theRNA molecule and the target molecule in the absence of the testmolecule.

The nucleic acid molecules may be derived from any ribonucleic acid,such as messenger RNA (mRNA), transfer RNA (tRNA), mitochondrial RNA(mtRNA), ribosomal RNA (rRNA), and nuclear RNA (nRNA). In anotheraspect, the ribonucleic acid molecules may comprise fragments of anysuch ribonucleic acids. For example, tRNA generally contains four stemloop structures: the TΨC-loop, the D-loop, a variable loop and theanticodon stem-loop. In addition to the four stem loop structures, thetRNA contains an acceptor arm for attachment of a particular amino acidfor incorporation into a peptide chain. The nucleic acid molecule foruse in the methods disclosed herein may comprise any one of the loopstructures of a tRNA. For example, a tRNA TΨC-loop fragment refers to anucleic acid sequence derived from, or corresponding to, the TΨC-loopsequence of a tRNA and includes at least part of the antiparallel stemstructure of the loop. In addition, the RNA molecule may consistessentially of one of the loop structures and include additional nucleicacid sequences not derived from, or not corresponding to, the samenative RNA molecule and may include detectable labels for use in themethods. The RNA molecule may also consist essentially of one of theloop structures in addition to one additional loop structure derivedfrom, or corresponding to, the same native RNA molecule, and may containadditional nucleotides or labels. For example, the ribonucleic acidsequence may comprise an anticodon stem-loop sequence, a TΨC-loopsequence, or a D-loop sequence. A ribonucleic acid molecule may alsocomprise a TΨC-loop sequence in combination with an anticodon stem loopsequence. In addition, the molecule may further comprise the acceptorarm of the tRNA molecule.

The nucleic acid molecules may contain any number of modified orunmodified nucleotides. In one aspect, the ribonucleic acid moleculesincorporate one, two, three, or more modified nucleotides into thenucleic acid sequence. Where two or more modified nucleotides areincorporated into a nucleic acid molecule, the modified nucleotides maybe the same or may be different modified nucleotides. In another aspect,the nucleic acid molecules incorporate three or more modifiednucleotides into the nucleic acid molecule.

The nucleic acid molecules containing the modified nucleotides may belinear or may form secondary or tertiary structures, such as loopstructures, bulges, pseudoknots, or turns. Nucleic acid molecules mayform secondary or tertiary structures for any reason, such as, forexample, stem-loop structures by the inclusion of complementarynucleotides at the 5′ and 3′ terminal ends of the molecule. Nucleic acidmolecules having secondary or tertiary structures may be employed in themethods discloses herein depending on the target molecule to which thenucleic acid molecule interacts.

The ribonucleic acid molecules may have any number of nucleotides, suchas, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 100, or more nucleotides. In one aspect, the ribonucleicacid molecule is derived from a nucleic acid sequence of a tRNA. Wherethe nucleic acid molecule is derived from a tRNA, the tRNA may containmodified nucleotides within the sequence corresponding to one or more ofthe loop structures, such as the TΨC-loop, the D-loop, or the anticodonstem loop (ASL). In one aspect, the modifications may correspond topositions 34, 37, and 39 in the anticodon stem loop of a tRNA. Inanother aspect, the modified nucleotides may correspond to positions 53,54, and 57 in the TΨC-loop of a tRNA. In a further aspect, themodifications may correspond to positions 16 and 19 in the D-loop of atRNA. In another aspect, the modifications may correspond tocombinations of any of these positions in the anticodon stem loop, theTΨC-loop, or the D-loop. The position numbers used herein refer to thenucleotide position numbering of the conventional tRNA numbering asdisclosed in Sprinzl, et al. Nucl. Acids. Res., 26, 148-153 (1998).

A tRNA fragment may comprise a nucleic acid molecule derived from orcorresponding to an anticodon stem loop (ASL) of a tRNA molecule. In oneaspect, a tRNA fragment derived from or corresponding to an ASLcomprises the nucleic acid sequence: 5′-a′b′c′d′e′NUXNNYNe″(orZ)d″c″b″a″; where X, Y, and Z refer to modified nucleotides, a′, b′, c′,d′, e′, a″, b″, c″, d″, and e″ refer to any nucleotide and a′, b′, c′,d′, and e′ are complementary to a″, b″, c″, d″, and e″, respectively,and N refers to any nucleotide. In one aspect, X refers to mnm5s2U, s2U,mnm5U, and mcm5s2U; Y refers to t6A, m2A, and ms2t6A, and Z refers to Ψ.

In another aspect, the tRNA fragment comprises a nucleic acid moleculederived from or corresponding to the TΨC-loop of a tRNA molecule. In oneaspect, a tRNA fragment derived from or corresponding to TΨC-loopcomprises the nucleic acid sequence5′-h′i′j′k′l′(ribothymidine)(Psi)CN(m1A)NNl″k″j″i″h″, where h′, i′, j′,k′, l′, h″, i″, j″, k″, and l″ refer to any nucleotide and h′, i′, j′,k′, and l′ are complementary to h″, i″, j″, k″, and l″, respectively,and N refers to any nucleotide.

In another aspect, the tRNA fragment may comprise a nucleic acidmolecule derived from or corresponding to the D-loop of a tRNA molecule.In one aspect, a tRNA fragment derived from or corresponding to a D-loopcomprises the nucleic acid sequence5′-m′n′o′p′NN(dihydrouridine)NN(dihydrouridine)NNp″o″n″m″, where m′, n′,o′, p′, m″, n″, o″, and p″ are any nucleotide and m′, n′, o′, and p′ arecomplementary to m″, n″, o″, and p″, respectively, and N refers to anynucleotide.

In another aspect, the tRNA fragment comprises a nucleic acid moleculederived from or corresponding to the ASL of a tRNA molecule. In oneaspect, a tRNA fragment derived from or corresponding to the ASLcomprises the nucleic acid sequence5′-N¹N²N³N¹4(mcm5s2U)N⁶N¹7(ms2t6A)N¹9(pseudouridine)N¹¹N¹², where N¹,N², N³, N⁴, N⁶, N⁷, N⁹, N¹¹, and N¹² refer to any nucleotide. Such ASLfragments may be linear fragments.

The tRNA fragments (or “probe tRNA fragments”) for use in the methods ofthe present disclosure can be a fragment derived from any tRNA. The tRNAfragment may be obtained or derived from or corresponds to a tRNA^(Ala),tRNA^(Arg), tRNA^(Asn), tRNA^(Asp), tRNA^(Cys), tRNA^(Gln), tRNA^(Glu),tRNA^(Gly), tRNA^(His), tRNA^(Ile), tRNA^(Leu), tRNA^(Lys), tRNA^(Met),tRNA^(Phe), tRNA^(Pro), tRNA^(Ser), tRNA^(Thr), tRNA^(Trp), tRNA^(Tyr),or tRNA^(Val). In one aspect, the tRNA fragment corresponds totRNA^(Lys). In another aspect, the tRNA fragment is derived from orcorresponds to the tRNA^(Lys) anticodon stem loop (ASL). In anotheraspect, the tRNA fragment corresponds to a fragment consisting ofnucleotides 32-43 of a bacterial tRNA^(Lys). In a further aspect, thetRNA fragment corresponds to a fragment consisting of nucleotides 32-43of the human tRNA^(Lys). The position numbers used herein refer to thenucleotide position numbering of the conventional tRNA numbering asdisclosed in Sprinzl, et al. Nucl. Acids. Res., 26, 148-153 (1998). Inone aspect, the tRNA fragment is a fragment from a host cell tRNA, suchas a mammalian host cell, including, but not limited to, human, feline,and simian host cells.

The tRNA fragment may correspond to any portion of the tRNA involved ininteracting, directly or indirectly, to the target molecule. In oneaspect, the tRNA fragment corresponds to the TΨC-loop, the D-loop or theanticodon stem loop (ASL) of the tRNA, or combinations thereof.

The tRNA fragment may correspond to any portion of the host cell's tRNAinvolved in nucleotide binding, such as the portion of the tRNA involvedin the reverse transcription (RT) complex formation. For example, thetRNA may be involved in binding to a retroviral genome to initiate,prime, or facilitate reverse transcription of the retroviral genome. Inone aspect, the fragment tRNA corresponds to a fragment of the anticodonstem loop of any tRNA. In one aspect, the fragment corresponds to afragment from the anticodon stem loop of tRNA-Lys. In another aspect,the tRNA fragment corresponds to a fragment from the anticodon stem loopof human tRNA-Lys. In another aspect, the tRNA fragment corresponds to afragment from nucleotides 32-43 of human tRNA-Lys3.

In another aspect, the tRNA fragments correspond to a portion of abacterial host cell tRNA involved in protein synthesis in the bacterialhost cell. For example, the tRNA fragment may be derived from aTΨC-loop, the D-loop or ASL from a bacterial tRNA that binds tobacterial molecules involved in protein synthesis. Bacterial moleculesinvolved in protein synthesis include, but are not limited to aminoacyltRNA synthetases, methyl transferases, pseudouridine synthases, tRNAsulfurtransferase, tRNA thiolase (ThiI), tRNA-guanine transglycosylase,ribosomal RNA (e.g. 5S ribosomal RNA), and transfer RNA. Each of theTΨC-loop, the D-loop or ASL may be involved in the binding of suchmolecules and may be important or required for protein synthesis in abacterial host. In one aspect, test compounds that inhibit the bindingof the bacterial tRNA to molecules involved in protein synthesis do notinhibit the binding a mammalian tRNAs to molecules involved in mammalianprotein synthesis.

The tRNA fragment may also be any length of a fragment from a tRNA. Inone aspect, the tRNA fragment comprises a fragment of between 5 to 25continuous nucleotides of a tRNA, 7 to 20 continuous nucleotides of atRNA, or between 10 to 20 continuous nucleotides of a tRNA. In anotheraspect, the fragment is a fragment of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18 continuous nucleotides of a tRNA. In a further aspect, thefragment is a fragment of 12 continuous nucleotides of a tRNA ASL.

In some aspects, the tRNA ASL fragment may or may not be capable offorming a secondary structure. In a one aspect, the tRNA ASL fragment isnot capable of forming a stem loop structure with itself. In anotheraspect, the fragment is a linear fragment of a tRNA that is not capableof forming a stem loop structure with itself.

The tRNA fragment may also be linked to additional nucleic acids. Forexample, a tRNA fragment may be linked to one or more additional nucleicacids depending on the assay method. In one aspect, the tRNA fragmentmay be linked to nucleotides used to attach the fragment to a solidsupport surface. In another aspect, the fragment tRNA is linked toadditional nucleic acid molecules at one or both terminal ends of thetRNA fragment. In another aspect, the fragment tRNA is linked toadditional nucleic acid molecules at both terminal ends. The additionalnucleic acid sequences can be any length, preferably between 8 and 16nucleotides, between 10 and 14 nucleotides, more preferably 12nucleotides in length. In one aspect, the terminal sequences do notallow the tRNA fragment to form a secondary structure, such as a loopstructure.

A variety of methods are known in the art for making nucleic acidshaving a particular sequence or that contain particular nucleic acidbases, sugars, internucleotide linkages, chemical moieties, and othercompositions and characteristics. Any one or any combination of thesemethods can be used to make a nucleic acid, polynucleotide, oroligonucleotide for the present invention. Said methods include, but arenot limited to: (1) chemical synthesis (usually, but not always, using anucleic acid synthesizer instrument); (2) post-synthesis chemicalmodification or derivatization; (3) cloning of a naturally occurring orsynthetic nucleic acid in a nucleic acid cloning vector (e.g., seeSambrook, et al., Molecular Cloning: A Laboratory Approach 2^(nd) ed.,Cold Spring Harbor Laboratory Press, 1989) such as, but not limited to aplasmid, bacteriophage (e.g., m13 or lamda), phagemid, cosmid, fosmid,YAC, or BAC cloning vector, including vectors for producingsingle-stranded DNA; (4) primer extension using an enzyme with DNAtemplate-dependent DNA polymerase activity, such as, but not limited to,Klenow, T4, T7, rBst, Taq, Tfl, or Tth DNA polymerases, includingmutated, truncated (e.g., exo-minus), or chemically-modified forms ofsuch enzymes; (5) PCR (e.g., see Dieffenbach, C. W., and Dveksler, eds.,PCR Primer: A Laboratory Manual, 1995, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.); (6) reverse transcription (includingboth isothermal synthesis and RT-PCR) using an enzyme with reversetranscriptase activity, such as, but not limited to, reversetranscriptases derived from avian myeloblasosis virus (AMV), Maloneymurine leukemia virus (MMLV), Bacillus stearothermophilus (rBst),Thermus thermophilus (Tth); (7) in vitro transcription using an enzymewith RNA polymerase activity, such as, but not limited to, SP6, T3, orT7 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, oranother enzyme; (8) use of restriction enzymes and/or modifying enzymes,including, but not limited to exo- or endonucleases, kinases, ligases,phosphatases, methylases, glycosylases, terminal transferases, includingkits containing such modifying enzymes and other reagents for makingparticular modifications in nucleic acids; (9) use of polynucleotidephosphorylases to make new randomized nucleic acids; (10) othercompositions, such as, but not limited to, a ribozyme ligase to join RNAmolecules; and/or (11) any combination of any of the above or othertechniques known in the art. Oligonucleotides and polynucleotides,including chimeric (i.e., composite) molecules and oligonucleotides withnon-naturally-occurring bases, sugars, and internucleotide linkages arecommercially available (e.g., see the 2000 Product and Service Catalog,TriLink Biotechnologies, San Diego, Calif., USA)

The RNA molecule or the target molecule, or both the RNA molecule andthe target molecule may be labeled to facilitate detection. In oneaspect, the RNA molecule is labeled with a fluorophore to facilitatedetection. In another aspect, the target molecule is labeled with biotinto facilitate detection. In yet another aspect, the RNA molecule islabeled with a fluorophore and the target molecule is labeled withbiotin.

In another aspect, the ribonucleic acid molecules having one or moremodified nucleic acids may be immobilized on a substrate to create anarray. An “array” may comprise a solid support with nucleic acid probesattached to the support. Arrays may comprise a plurality of differentnucleic acids that are coupled to a surface of a substrate in different,known locations. These arrays, also described as “microarrays” orcolloquially “chips” have been generally described in the art, forexample, in Fodor et al., Science, 251:767-777 (1991). Methods offorming high density arrays of oligonucleotides, peptides and otherpolymer sequences with a minimal number of synthetic steps are disclosedin, for example, U.S. Pat. Nos. 5,143,854, 5,252,743, 5,384,261,5,405,783, 5,424,186, 5,429,807, 5,445,943, 5,510,270, 5,677,195,5,571,639, and 6,040,138. The oligonucleotide analogue array can besynthesized on a solid substrate by a variety of methods, including, butnot limited to, light-directed chemical coupling, and mechanicallydirected coupling. See Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCT Publication Nos.WO 92/10092 and WO 93/09668, U.S. Pat. Nos. 5,677,195, 5,800,992 and6,156,501 which disclose methods of forming vast arrays of peptides,oligonucleotides and other molecules using, for example, light-directedsynthesis techniques. See also, Fodor et al., Science, 251, 767-77(1991). These procedures for synthesis of polymer arrays are nowreferred to as VLSIPS® procedures. Using the VLSIPS®. approach, oneheterogeneous array of polymers is converted, through simultaneouscoupling at a number of reaction sites, into a different heterogeneousarray. See, U.S. Pat. Nos. 5,384,261 and 5,677,195.

Methods for making and using molecular probe arrays (particularlynucleic acid probe arrays) are also disclosed in, for example, U.S. Pat.Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,5,409,810, 5,412,087, 5,424,186, 5,429,807, 5,445,934, 5,451,683,5,482,867, 5,489,678, 5,491,074, 5,510,270, 5,527,681, 5,527,681,5,541,061, 5,550,215, 5,554,501, 5,556,752, 5,556,961, 5,571,639,5,583,211, 5,593,839, 5,599,695, 5,607,832, 5,624,711, 5,677,195,5,744,101, 5,744,305, 5,753,788, 5,770,456, 5,770,722, 5,831,070,5,856,101, 5,885,837, 5,889,165, 5,919,523, 5,922,591, 5,925,517,5,658,734, 6,022,963, 6,150,147, 6,147,205, 6,153,743, 6,140,044 andD430024. In one aspect, the ribonucleic acid molecule is a labeled witha detectable label, such as a fluorescent label. A sample containing atarget molecule is contacted with the aay under appropriate conditions.The arrays are washed or otherwise processed to remove non-boundmolecules. The reaction is then evaluated by detecting the distributionof the label on the chip. The distribution of label may be detected byscanning the arrays to determine florescence intensities distribution.Methods for signal detection and processing of intensity data aredisclosed in, for example, U.S. Pat. Nos. 5,547,839, 5,578,832,5,631,734, 5,800,992, 5,856,092, 5,936,324, 5,981,956, 6,025,601,6,090,555, 6,141,096, and 5,902,723. Methods for array based assays,computer software for data analysis and applications are disclosed in,for example, U.S. Pat. Nos. 5,527,670, 5,527,676, 5,545,531, 5,622,829,5,631,128, 5,639,423, 5,646,039, 5,650,268, 5,654,155, 5,674,742,5,710,000, 5,733,729, 5,795,716, 5,814,450, 5,821,328, 5,824,477,5,834,252, 5,834,758, 5,837,832, 5,843,655, 5,856,086, 5,856,104,5,856,174, 5,858,659, 5,861,242, 5,869,244, 5,871,928, 5,874,219,5,902,723, 5,925,525, 5,928,905, 5,935,793, 5,945,334, 5,959,098,5,968,730, 5,968,740, 5,974,164, 5,981,174, 5,981,185, 5,985,651,6,013,440, 6,013,449, 6,020,135, 6,027,880, 6,027,894, 6,033,850,6,033,860, 6,037,124, 6,040,138, 6,040,193, 6,043,080, 6,045,996,6,050,719, 6,066,454, 6,083,697, 6,114,116, 6,114,122, 6,121,048,6,124,102, 6,130,046, 6,132,580, 6,132,996 and 6,136,269.

The use of high-density arrays allows for the development of highthroughput assays for the disclosed methods. Such high throughput (HTS)assays may involve attaching or binding either the ribonucleic acidmolecule or the target molecule to a solid support. A “solid support”may be any surface to which molecules may be attached through eithercovalent or non-covalent bonds. This includes, but is not limited to,membranes, plastics, magnetic beads, charged paper, nylon,Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE,polystyrene, gallium arsenide, gold, and silver. Any other materialknown in the art that is capable of having functional groups such asamino, carboxyl, thiol or hydroxyl incorporated on its surface, is alsocontemplated. This includes surfaces with any topology, including, butnot limited to, spherical surfaces and grooved surfaces. HTS methodsgenerally refer to technologies that permit the rapid assaying of testcompounds for therapeutic potential, for example, by inhibiting thebinding of a tRNA fragment to a target nucleic acid molecule. HTStechniques may employ robotic handling of test materials, detection ofpositive signals, and interpretation of data. Test compounds may beidentified via the detection of luminescence or absence of luminescencethrough the use of radioactivity or through optical assays that rely onabsorbence, fluorescence or luminescence as read-outs. Gonzalez, J. E.et al., (1998) Curr. Opin. Biotech. 9:624-631.

Such microarrays containing the ribonucleic acid molecules of thepresent disclosure may be used in combination with any of the methodsdisclosed herein for the development of high-throughput assays. Nucleicacid molecules for use in developing such microarrays may be identifiedby first analyzing databases of various genomes and nucleotidemodifications to select desired sequences. Modified nucleotides are thenprepared using proper protection for automated chemical synthesis.Nucleic acid molecules are synthesized incorporating the modifiednucleotides. The nucleic acid molecules are synthesized either attachedto a solid support surface or in solution. Oligonucleotides prepared forsolution array will have fluorescent labels attached to monitorinteraction or binding with target molecules. Target molecules areprepared by appropriate methods. For solid phase arrays, the targetmolecule will have fluorescent labels attached, while no labeling isneeded for solution based assays. The arrays are then interrogated withthe target molecule solutions to identify nucleic acid molecules havingthe highest affinity for the target molecules.

The ribonucleic acid molecules may be used in methods of identifying aninhibitor of nucleic acid-target molecule interactions. The methods canbe used to identify any inhibitor of such interactions. In one aspect,the method can be used to identify inhibitors of RNA-target moleculebinding. In one aspect, the methods can be used to identify inhibitorsof protein synthesis. In another aspect, the methods can be used toidentify inhibitors of tRNA binding to a target nucleic acid molecule.In another aspect, the methods can be used to identify inhibitors oftRNA binding to a target aminoacyl RNA synthetase. In a further aspect,the methods can be used to identify inhibitors of tRNA binding to atarget ribosome. In another aspect, the methods can be used to identifyinhibitors of tRNA modification enzymes. In another aspect, the methodscan be used to identify inhibitors of tRNA binding to a target viralreverse transcriptase.

In another aspect, the methods can be readily adapted for use inhigh-throughput assays using high-density arrays having one or more RNAprobe molecules attached. Transfer RNA (tRNA) is capable of interactingwith numerous biological molecules such as those involved in proteinsynthesis, transcription, translation, reverse transcription, and thelike. Identifying inhibitors of such interactions may lead to theidentification of therapeutic compounds for use in treating diseases orinfections in a host cell. In one aspect, the target molecule isessential to protein synthesis. In one aspect, compounds that inhibitthe binding of the bacterial tRNA to molecules involved in proteinsynthesis do not inhibit the binding a mammalian tRNAs to moleculesinvolved in mammalian protein synthesis.

The inhibitors that are identified by the disclosed methods may beuseful for treating any infectious disease or condition in a host cell,such as, for example, bacterial diseases, viral diseases, includingretroviral diseases, protozoan diseases, and fungal diseases.

Bacterial diseases for which inhibitors can be identified by the methodsdisclosed herein include any bacterial disease, such as infectionscaused by, for example, category A, B, or C pathogens. Category Abacteria include, but are not limited to, Bacillus antracis, Clostridiumbotulinum, Francisella tularensis, and Yersinia pestis. Category Bbacteria include, but are not limited to Burkholderia pseudomallei,Burkjolderia mallei, Clostridium perfringens, Coxiella burnetii,Brucella melitensis, Brucella abortus, Brucella canis, Staphylococcusaureus, Rickettsia prowazekii, Chlamydia psittaci, and food and waterborne bacteria, such as Escherichia coli O157:H7.

Viral Diseases for which inhibitors can be identified by any of themethods disclosed herein include, for example, retroviruses (includinglentivirus) and other viral diseases, such as viral encephalitis.

Retroviruses for which inhibitors can be identified by the methodsdisclosed herein include any viruses having RNA as their primary geneticmaterial and use reverse transcription to produce DNA. Such virusesinclude, but are not limited to, Feline Immunodeficiency Virus (FIV),Simian Immunodeficiency Virus (SIV), Avian Leucosis Virus, FelineLeukemia Virus, Walleye Dermal Sarcoma Virus, Human T-LymphotropicVirus, and Human Immunodeficiency Viruses (HIV). HIV can be any strain,form, subtype or variation in the HIV family.

The target molecules involved in protein synthesis in pathogen cells maybind to any portion of a tRNA molecule, for example the TΨC-loop, theD-loop, and/or the anticodon stem loop. For example, target moleculessuch as aminoacyl synthetases, methyl transferases and pseudouridinesynthases may bind to the pathogen's TΨC-loop, D-loop or ASL orcombinations thereof to initiate the particular protein reaction. Apartial list of target molecules, their function and related pathogenare provided in Table 1.

TABLE 1 Target Molecule Function Gene Product Pathogen Protein AminoacyltRNA Essential Bacterial/Fungal Synthetase Protein Methyl transferaseEssential Bacterial/Fungal Protein Pseudouridine EssentialBacterial/Fungal synthase Protein tRNA Essential Bacterial/Fungalsulfurtransferase Protein tRNA thiolase Essential Bacterial/Fungal(ThiI) Protein tRNA-guanine Essential in Bacterial/Fungaltransglycosylase some pathogens Nucleic Acid 5 S Ribosomal RNA EssentialBacterial/Fungal (RNA) Nucleic Acid Transfer RNA EssentialBacterial/Fungal (RNA) Nucleic Acid Viral RNA Non-EssentialLentiviruses, (RNA) HCV

Thus, the disclosure provides a method of identifying an inhibitor oftRNA-target molecule interactions. The method comprises forming amixture of a nucleic acid molecule comprising a tRNA TΨC-loop fragmenthaving at least one modified nucleotide, a target molecule that iscapable of binding to the tRNA TΨC-loop fragment, and a test compound.The resulting mixture is incubated under conditions that allow bindingof the tRNA TΨC-loop fragment and the target molecule in the absence ofthe test compound. The method further involves detecting whether thetest compound inhibits the binding of the tRNA TΨC-loop fragment to thetarget molecule, where the absence of binding of the tRNA TΨC-loopfragment and the target molecule is indicative of the test compoundbeing an inhibitor of tRNA-target molecule interaction. In one aspect,the detection involves the use of labels to detect the inhibition ofbinding of the tRNA fragment to a target molecule.

The mixture may also contain a second nucleic acid molecule thatcontains an anticodon stem-loop sequence. The second nucleic acidmolecule may also contain one or more modified nucleotides. The secondnucleic acid molecule may be linked to the first nucleic acid moleculeand may facilitate binding of the TΨC-loop fragment to the targetmolecule in the absence of the test compound.

The nucleic acid molecule may be attached to a microarray to provide fora high-throughput assay for use in identifying such inhibitors.

In one aspect, a method of identifying an inhibitor of tRNA-targetmolecule interactions is also provided, where the method comprisesforming a mixture of a nucleic acid molecule comprising a tRNA D-loopfragment having at least one modified nucleotide, a target molecule thatis capable of binding to the tRNA D-loop fragment, and a test compound.The resulting mixture is incubated under conditions that allow bindingof the tRNA D-loop fragment and the target molecule in the absence ofthe test compound. The method further involves detecting whether thetest compound inhibits the binding of the tRNA D-loop fragment to thetarget molecule, where the absence of binding of the tRNA D-loopfragment and the target molecule is indicative of the test compoundbeing an inhibitor of tRNA-target molecule interaction. In one aspect,the detection involves the use of labels to detect the inhibition ofbinding of the tRNA fragment to a target molecule.

In one aspect, a method of identifying an inhibitor of binding of a tRNAto a target nucleic acid molecule comprises forming a mixture containinga tRNA ASL fragment, a target nucleic acid molecule that is capable ofbinding to the tRNA fragment, and a test compound. The resulting mixtureis incubated under conditions that allow binding of the tRNA fragmentand the target nucleic acid in the absence of the test compound. Themethod further involves detecting whether the test compound inhibits thebinding of the tRNA fragment to the target nucleic acid, where bindingof the tRNA ASL fragment and the target nucleic acid molecule isindicative of the test compound being an inhibitor of binding of a tRNAto a target nucleic acid molecule. In one aspect, the detection involvesthe use of labels to detect the inhibition of binding of the tRNAfragment to the target nucleic acid molecule.

The disclosure also provides a method of identifying an inhibitor oftRNA-target molecule interactions, where the method comprises forming amixture of a nucleic acid molecule comprising a tRNA D-loop fragmenthaving at least one modified nucleotide, a tRNA ASL fragment, a targetmolecule that is capable of binding to the tRNA D-loop and/or ASLfragment, and a test compound. The tRNA ASL fragment may also containone or more modified nucleotides. The resulting mixture is incubatedunder conditions that allow binding of the tRNA D-loop fragment and/orthe ASL fragment and the target molecule in the absence of the testcompound. The method further involves detecting whether the testcompound inhibits the binding of the tRNA D-loop fragment and/or ASLfragment to the target molecule, where the absence of binding of thetRNA D-loop fragment and/or ASL fragment and the target molecule isindicative of the test compound being an inhibitor of tRNA-targetmolecule interaction. In one aspect, the detection involves the use oflabels to detect the inhibition of binding of the tRNA fragment to atarget molecule.

In one aspect, the tRNA D-loop fragment and the ASL fragment may becombined in a single RNA molecule, or may be used as separate nucleicacid molecules in the mixture. Where the D-loop fragment and the ASLfragment are contained in a single RNA molecule, the D-loop and the ASLmay be in any arrangement, including the naturally occurring arrangementand a non-naturally occurring arrangement.

In one aspect, a method of identifying an inhibitor of tRNA-targetmolecule interactions comprises forming a mixture of a nucleic acidmolecule comprising a tRNA TΨC-loop fragment having at least onemodified nucleotide, a tRNA ASL fragment, a target molecule that iscapable of binding to the tRNA TΨC-loop fragment and/or the ASLfragment, and a test compound. The resulting mixture is incubated underconditions that allow binding of the tRNA TΨC-loop fragment and/or theASL fragment and the target molecule in the absence of the testcompound. The method further involves detecting whether the testcompound inhibits the binding of the tRNA TΨC-loop fragment and/or ASLfragment to the target molecule, where the absence of binding of thetRNA TΨC-loop fragment and/or ASL fragment and the target molecule isindicative of the test compound being an inhibitor of tRNA-targetmolecule interaction. In one aspect, the detection involves the use oflabels to detect the inhibition of binding of the tRNA fragment to atarget molecule.

In one aspect, the tRNA TΨC-loop fragment and the ASL fragment may becombined in a single RNA molecule, or may be used as separate nucleicacid molecules in the mixture. Where the TΨC-loop fragment and the ASLfragment are contained in a single RNA molecule, the TΨC-loop and theASL may be in any arrangement, including the naturally occurringarrangement and a non-naturally occurring arrangement.

In one aspect, a method of identifying an inhibitor of tRNA-targetmolecule interactions comprises forming a mixture of a nucleic acidmolecule comprising a tRNA TΨC-loop fragment having at least onemodified nucleotide, a D-loop fragment, and a tRNA ASL fragment, atarget molecule that is capable of binding to the tRNA TΨC-loop fragmentand/or D-loop fragment and/or the ASL fragment, and a test compound. Theresulting mixture is incubated under conditions that allow binding ofthe tRNA TΨC-loop fragment and/or D-loop fragment and/or the ASLfragment and the target molecule in the absence of the test compound.The method further involves detecting whether the test compound inhibitsthe binding of the tRNA TΨC-loop fragment and/or D-loop fragment and/orASL fragment to the target molecule, where the absence of binding of thetRNA TΨC-loop fragment and/or D-loop fragment and/or ASL fragment andthe target molecule is indicative of the test compound being aninhibitor of tRNA-target molecule interaction. In one aspect, thedetection involves the use of labels to detect the inhibition of bindingof the tRNA fragment to a target molecule. The TΨC-loop fragment, D-loopfragment and ASL may be in separate nucleic acid molecules, or may belinked on a single nucleic acid molecule, and may in any arrangement,such as the naturally occurring arrangement or non-naturally occurringarrangement.

The disclosure also provides methods for identifying inhibitors ofCategory B bacteria infection. Such methods comprise forming a mixturecontaining a tRNA fragment from a Category B Bacteria having one or moremodified nucleotide bases, a target molecule from a Category B Bacteriacapable of binding to the tRNA fragment, and a test compound. Theresulting mixture is incubated under conditions that allow binding ofthe tRNA fragment and the target molecule in the absence of the testcompound. The method further involves detecting whether the testcompound inhibits the binding of the tRNA fragment to the targetmolecule. In one aspect, the detection involves the use of labels todetect the inhibition of binding of the tRNA fragment to the targetmolecule, where the inhibition indicates that the test compound iscapable of inhibiting infection of a host cell by category B bacteria.The target molecule from a Category B Bacteria may be an aminoacyl tRNAsynthetase, a methyl transferase, a pseudouridine synthase, a ribosomalRNA (e.g. 5S ribosomal RNA), or a transfer RNA.

In another aspect, the disclosure provides methods for identifyinginhibitors of aminoacyl-tRNA synthetases (AaRS) for use as antimicrobialagents. In one aspect, criteria for such inhibitors also include: i)diversity between prokaryotic and eukaryotic sequences to developinhibitors that selectively target the prokaryote; ii) conserved targetsacross different pathogens for development of broad spectrumantimicrobial compounds; iii) 20 potential targets as each amino acidhas its own AaRS; iv) target molecules that are soluble, stable, andrelatively easy to purify for use in an HTS; and v) X-ray crystalstructures. In another aspect, the target molecule may be an essentialgene for bacterial growth and/or propagation. For bacterial pathogensmodified nucleotides at position 34 of the tRNA may be important fortRNA-AaRS binding. For example, in GluRS binding, a modified nucleotideat position 34 may be important for efficient charging of the tRNA inbacteria for binding to the synthetase.

In one aspect, the AaRS is LysRS. In addition to this being an essentialenzyme, LysRS from bacteria utilizes a different modified nucleotidebase and RNA sequence in the anticodon stem loop than mammals. LysRS hasbeen isolated and purified from many species indicating that this enzymeis soluble, stable, and relatively easy to isolate. The crystalstructure of LysRS has been solved for several bacteria. In addition,enzyme homology of LysRS of human to the food and water borne bacteriais less than 10% (FIGS. 1A-D, Table 2). The homology for LysRS among thebacteria may range from approximately 20% to nearly 100%. Thus, in oneaspect, inhibitors identified by these methods inhibit the binding ofbacterial AaRS to the bacterial tRNA, but does not inhibit the mammalianAaRS to the mammalian tRNA.

TABLE 2 Shi E C Sal Yer Lis Cam Vib H S Shigella 1.00 0.99 0.95 0.86 0.50.46 0.19 0.08 flexneri Escherichia 0.99 1.00 0.95 0.86 0.5 0.46 0.190.08 coli Salmonella 0.95 0.95 1.00 0.85 0.5 0.45 0.18 0.08 entericaYersinia 0.86 0.86 0.85 1.00 0.48 0.47 0.19 0.07 enterocolitica Listeria0.5 0.5 0.5 0.48 1.00 0.45 0.2 0.09 monocytogenes Campylobacter 0.460.46 0.45 0.47 0.45 1.00 0.19 0.07 jejuni Vibrio 0.19 0.19 0.18 0.19 0.20.19 1.00 0.07 cholerae Homo sapiens 0.08 0.08 0.08 0.07 0.09 0.07 0.071.00

In another aspect, the methods may involve the detection of the bindingof the test compound to the tRNA fragment, the target molecule, or boththe tRNA fragment and the target molecule. In one aspect, the binding ofthe test compound is indicative of the test compound being an inhibitorof bacterial propagation, bacterial infection, protein synthesis, ortRNA binding.

In another aspect, methods for diagnosing (or assisting in diagnosing)whether a subject has an infectious disease are also provided. Suchmethods may be used to diagnose (or assist in diagnosis) of anyinfectious disease, including, but not limited to bacterial diseases,viral diseases, and fungal diseases.

Thus, in one aspect, a method for diagnosing (or assisting indiagnosing) whether a subject has a bacterial infection is provided. Themethod comprises forming a mixture comprising a first RNA moleculehaving at least one modified nucleotide and a biological sample obtainedfrom a subject. The mixture is then incubated under conditions thatallow the interaction, or binding, of the first RNA molecule and atarget molecule in the biological sample, then detecting whether or notthe first RNA molecule binds to the target molecule in the biologicalsample. Binding of the first RNA molecule and the target molecule isindicative of the positive diagnosis of a bacterial infection. The lackof binding of the RNA molecule and the target molecule is indicative ofthe negative diagnosis of a bacterial infection.

In another aspect, a method for identifying target molecules that bindto nucleic acid molecules having one or more modified nucleotides isprovided. The methods generally comprise forming a mixture of abiological sample from a subject and at least one nucleic acid moleculederived from, or corresponding to, a tRNA loop having at least onemodified nucleotide. The mixture is incubated under conditions thatallow binding of a target molecule in the biological sample to the firstnucleic acid molecule. After the incubation, target molecules that bindto the nucleic acid molecule can be detected. A washing step may also beused to remove unbound molecules and facilitate the detection of thebound molecules. The tRNA loop molecules that can be used in the mixtureinclude those selected from the group consisting of a TΨC-loop, aD-loop, and an anticodon loop, and combinations thereof.

The tRNA loop molecules may be derived from or correspond to tRNAsequences from any organism. For example, tRNA loop molecules for use inthe methods may be derived from, or correspond to, tRNA sequences frombacterial, fungal, or viral pathogens, and may incorporate modifiednucleotides based on the particular pathogen.

The nucleic acid molecules may be arranged on an array to allow for thedevelopment of high throughput assays for the detection of moleculesthat bind to nucleic acid molecules having a modified nucleotide. Sucharrays may contain multiple nucleic acid molecules derived from tRNATΨC-loop fragments, D-loop fragments, and anticodon loop fragments, toallow for the simultaneous screening of one or more different nucleicacid sequences having one or more different modified nucleotides. Theone or more different nucleic acid sequences may be derived from orcorrespond to sequences from a single pathogen or a group of pathogensto identify target compounds for which broad spectrum inhibitors may beidentified. In addition, the nucleic acid molecules may be detectablylabeled to further facilitate detection of the binding of a targetmolecule to a tRNA molecule.

Such methods may be useful in the development of diagnostic methods todetect whether a subject is infected with a particular pathogen. Inaddition, molecules that are identified as binding to the tRNA loopmolecules by such methods can be used in inhibition assays as disclosedherein to identify compounds useful for the inhibition of pathogeninfection in a subject. Such compounds may be useful as therapeuticcompounds or in the development of therapeutic compounds for thetreatment of pathogen infections.

The methods for detecting binding of the target molecule to the tRNA orthe inhibition of such binding or the diagnostic methods may beperformed using any method for such detection, for example, theAlphaScreen® assay (Packard Instrument Company, Meriden, Conn.).AlphaScreen® technology is an “Amplified Luminescent ProximityHomogeneous Assay” method utilizing latex microbeads (250 nm diameter)containing a photosensitizer (donor beads), or chemiluminescent groupsand fluorescent acceptor molecules (acceptor beads). Upon illuminationwith laser light at 680 nm, the photosensitizer in the donor beadconverts ambient oxygen to singlet-state oxygen. The excitedsinglet-state oxygen molecules diffuse approximately 250 nm (one beaddiameter) before rapidly decaying. If the acceptor bead is in closeproximity to the donor bead (i.e., by virtue of the interaction of thetarget molecule and RNA molecule), the singlet-state oxygen moleculesreact with chemiluminescent groups in the acceptor beads, whichimmediately transfer energy to fluorescent acceptors in the same bead.These fluorescent acceptors shift the emission wavelength to 520-620 nm,resulting in a detectable signal. Antagonists of the interaction of thetarget molecule with the RNA molecule will thus inhibit the shift inemission wavelength, whereas agonists of this interaction would enhanceit.

The disclosed methods may be performed by mixing the componentnucleotides (e.g. the RNA molecule), the target molecule and the testcompound in any order, or simultaneously. For example, a target moleculemay be first combined with a test compound to form a first mixture, andthen an RNA molecule may be added to form a second mixture. In anotherexample, a target molecule, an RNA molecule and the test compound mayall be mixed at the same time before incubation. In one aspect, themixture is incubated under conditions that allow binding of the RNAmolecule and the target molecule in the absence of the test compound.

The inhibition of binding of the RNA molecule and the target molecule bythe test Compound may be detected using any method available for thedetection of inhibition. In one aspect, the determining step may beperformed using methods including, but not limited to, gel shift assays,chemical and enzymatic footprinting, circular dichroism and NMRspectroscopy, equilibrium dialysis, or in any of the binding detectionmechanisms commonly employed with combinatorial libraries of probes ortest compounds. The inhibition of binding indicates that the testcompound may be useful for inhibiting the interaction between an RNAmolecule and a target molecule.

Any compound may be tested using the methods of the present invention toidentify compounds capable of inhibiting interactions between an RNA anda target molecule. Test compounds that may be screened with methods ofthe present invention include, but are not limited to, polypeptides,beta-turn mimetics, polysaccharides, phospholipids, hormones,prostaglandins, steroids, aromatic compounds, heterocyclic compounds,benzodiazepines, oligomeric N-substituted glycines, oligocarbamates,polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Some testcompounds are synthetic molecules while others are natural molecules.

Test compounds may be obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. Combinatorial libraries canbe produced for many types of compound that can be synthesized in astep-by-step fashion. Large combinatorial libraries of compounds can beconstructed by the encoded synthetic libraries (ESL) method described inWO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642.Peptide libraries can also be generated by phage display methods (see,e.g., Devlin, WO 91/18980). Libraries of natural compounds in the formof bacterial, fungal, plant and animal extracts can be obtained fromcommercial sources or collected in the field. Known pharmacologicalagents can be subject to directed or random chemical modifications, suchas acylation, alkylation, esterification, amidification to producestructural analogs.

Combinatorial libraries of peptides or other compounds can be fullyrandomized, with no sequence preferences or constants at any position.Alternatively, the library can be biased, i.e., some positions withinthe sequence are either held constant, or are selected from a limitednumber of possibilities. For example, in some cases, the nucleotides oramino acid residues are randomized within a defined class, for example,of hydrophobic amino acids, hydrophilic residues, sterically biased(either small or large) residues, towards the creation of cysteines, forcross-linking, prolines for SH-3 domains, serines, threonines, tyrosinesor histidines for phosphorylation sites, or to purines.

In another aspect, the test compounds may be naturally occurringproteins or their fragments. Such test compounds may be obtained from anatural source, e.g., a cell or tissue lysate. Libraries of polypeptideagents may also be prepared, e.g., from a cDNA library commerciallyavailable or generated with routine methods. The test compounds can alsobe peptides, e.g., peptides of from about 5 to about 30 amino acids,with from about 5 to about 20 amino acids being preferred and from about7 to about 15 being particularly preferred. The peptides can be digestsof naturally occurring proteins, random peptides, or “biased” randompeptides. In some methods, the test compounds are polypeptides orproteins.

In another aspect, the test compounds may be nucleic acids. Nucleic acidtest compounds may be naturally occurring nucleic acids, random nucleicacids, or “biased” random nucleic acids. For example, digests ofprokaryotic or eukaryotic genomes may be similarly used as describedabove for proteins.

In some preferred methods, the test compounds are small molecules, e.g.,molecules with a molecular weight of not more than about 500 or 1,000.Preferably, high throughput assays are adapted and used to screen forsuch small molecules. In some methods, combinatorial libraries of smallmolecule test compounds as described above can be readily employed toscreen for small molecule modulators of retroviral propagation. A numberof assays are available for such screening, e.g., as described inSchultz et al., Bioorg Med Chem Lett 8:2409-2414, 1998; Weller et al.,Mol Divers. 3:61-70, 1997; Fernandes et al., Curr Opin Chem Biol2:597-603, 1998; and Sittampalam et al., Curr Opin Chem Biol 1:384-91,1997.

The invention also comprises kits and compositions (e.g., reactionmixtures, etc.) for a method of the invention. A kit is a combination ofindividual compositions useful or sufficient for carrying out one ormore steps of a method of the invention, wherein the compositions areoptimized for use together in the method. A composition comprises anindividual component for at least one step of a method of the invention.The present disclosure further provides a kit for screening for aninhibitor of interactions between an RNA molecule and a target molecule,comprising: an RNA fragment and a reagent for detection of binding to atarget molecule, such as a detectable label. In some embodiments, thekit also comprises one or more target molecule(s) that are capable ofbinding to the RNA fragment. In some embodiments, the kit furthercomprises additional reagents for conducting the screening methods. Insome embodiments, the kit further comprises a plurality of inhibitors ofretroviral reverse transcription. In some embodiments, the reagentcomprises a dye that undergoes fluorescence enhancement upon binding tonucleic acids (e.g., the dye is RIBOGREEN, SYBR Gold, SYBR Green I, orSYBER Green II). In some embodiments, the kit further comprises controlreagents (e.g., sample polymerases and/or inhibitors for positivecontrols, polymerase and/or inhibitor minus samples for negativecontrols, etc.). In some embodiments, the kit further comprisesinstructions for carryout out the methods. In some embodiments, theinstructions are embodied in computer software that assists the user inobtaining, analyzing, displaying, and/or storing results of the method.The software may further comprise instructions for managing sampleinformation, integrating with scientific equipment (e.g., detectionequipment), etc.

Also provided are kits for identifying inhibitors bacterial infection.In one aspect, the kits include, a tRNA fragment and a detectable labelfor detecting the interaction of the tRNA fragment and a target moleculefrom a biological sample. The kits of the present invention may alsoinclude target molecules, and/or reagents for performing the assays. Thekits may also include labeling components for detecting whether a testcompound inhibits the binding of the fragment tRNA and the targetmolecule.

Also provided are kits for diagnosing whether a subject is infected withan infectious disease. Such kits comprise: an RNA fragment and a reagentfor detection of binding to a target molecule from a biological sample(such as a detectable label).

The invention will be further explained by the following illustrativeexamples that are intended to be non-limiting.

EXAMPLES Example 1 Synthesis of RNA Sequences Having ModifiedNucleotides

The first step in producing the fragment RNA molecule sequences is thesynthesis of the modified nucleotides, also known as phosphoramidites(Agris et. al Biochimie. (1995) 77(1-2):125-34). The modifiednucleotides are then used during the synthesis of the RNA oligomers(Ogilvie et. al. Proc Natl Acad Sci USA. (1988) 85:5764-8). Syntheticapproaches overcome the substantial barrier of obtaining sufficientamounts of natural products for the functional characterization studies.In addition to providing the fully modified RNA for characterization ofthe fragment tRNA:target molecule binding, the synthetic approach allowsfor the preparation of intermediate steps/forms of the modified materialthat can further elucidate the individual contribution of eachmodification step in enhanced tRNA binding.

Modified base nucleic acid molecules were prepared using a combinationof methods for the synthesis, incorporation, and purification of all themodified nucleotides. Modified base phosphoramidites were prepared usingknown methods, such as those disclosed in Ogilive et. al., Proc. Natl.Acad. Sci., U.S.A., 85:5764-5768 (1988). S (mnm⁵s²U) was preparedfollowing the previously published procedures of Vorbruggen et. al.,Angew. Chem., 14: 225-256 (1975). 6 (t^(6A)) and $ (cmnm⁵s²U) weresynthesized following the procedures described below. Pseudouridine iscommercially available and was incorporated into the RNA oligomers usingthe methods described below. Functional groups on the modifiednucleotide bases were protected using phosphoramidite chemistry (Ogilvieet. al., 1988). Using this chemistry, over 20 different modifiednucleotides have been incorporated into a range of oligonucleotidesranging in length from 3 to 36 nucleotides (Nobles, et. al., Nuc. AcidsRes., 30: 4751-4760 (2002)). The addition of a protecting group to eachmodified base and ribose is described below. The protecting group wassubsequently removed after synthesis of the RNA oligomer. While 2position thio-groups can be oxidized in standard RNA synthesis protocolsthis barrier has been overcome by using the tert-butyl hydroperoxide(10% solution in acetonitrile) oxidizing agent (Kumar and Davis, Nuc.Acids Res., 25(6): 1272-1280 (1997)).

Compound 1—mnm⁵s²U Phosphoramidite

Base protection of mnm⁵s²U [5-(N-trifiluoroacetyl)methylaminomethy2-thiouridine]: The 2,2,2-trichloroethoxycarbonyl protecting groups wereused to protect exo-amino function of the nucleotide. The nucleotide istreated with excess of trifluoroacetic anhydride in pyridine solution,followed by selective removal of trifluoroacetyl groups from sugarmoiety with 10% sodium bicarbonate giving the base protected product(Malkiewicz, Tetrahedron Letters, 24: 5387-5390 (1983)).

Protection of the ribose and phosphitylation follow the general scheme:

Base Protection of mnm⁵s²U

Preparation of the N⁶—(N-threonylcarbonyl)adenosine for automatedsynthesis follows a slightly different approach than that for the otherphosphoramidites. First, the ribose functions of adenosine are protectedfollowing the methods outlined above. Next, the ribose protectedadenosine was reacted with 3 equivalents of phenoxycarbonyltetrazole inanhydrous dioxane for 18 hr at 37° C. to produce phenyl carbamates atthe six position. This was followed by aminolysis with 3 equivalents ofcrystalline L-threonine p-nitrobenzyl ester in anhydrous dioxane, for 18hr at 37° C., producing the N6-(N-threonylcarbonyl)adenosine. The t6Acarboxylate was then protected by a trimethylsilylethyl group, in amanner similar to that used to protect the ribose function. Finally thephosphoramidite was phosphitylated following the protocol describedbelow.

Modification of Adenosine to t6A

Preparation of the 3—cmnm⁵s²U nucleotide follows published methods(Reese and Sanghvi, J. Chem. Soc. Chem. Commun., 62-63 (1984)). Briefly,2 thiouridine was heated with 5 molar equivalents each of pyrrolidineand formaldehyde in aqueous solution for 1 h, under reflux to produce2′,3′-O-isopropylidene-5-pyrrolidinomethyl-2-thiouridine. This base wastreated with 10 mol. equivalents of methyl iodide in acetonitrile atroom temperature. After 16 hours, the products were concentrated underreduced pressure to give the putative niethiodide which was thendissolved in acetonitrile and allowed to react with 3 molar equivalentsof glycine t-butyl ester at room temperature for 16 h. This product wasthen purified and protection of the ribose and phosphitylation followthe general scheme:

Preparation and Base Protection of cmnm5s2U

General procedure for ribose protection and phosphitylation prior to RNAoligomer synthesis: After base protection the scheme for the synthesisof 5′-O-(4,4′-dimethoxytrityl)-2′-O-tertbutyldimethylsilyi-modifiedribonucleoside-3′-O-(2-cyanoethyl-N-diisopropyl)-phosphoramidites is thesame for all modified nucleotides. The protected nucleoside was dried byco-evaporation twice with pyridine and dissolved in pyridine.Tert-butyldimethylchlorosilane and imidazole were added and reacted for4 hours at room temperature. The excess silyl chloride was decomposedwith water and dichloromethane. The aqueous layer was extracted twicewith dichloromethane and combined with the organic layer. The solventwas evaporated by vacuum yielding a gum which was dissolved in ether andprecipitated by pouring slowly into petroleum ether (40-60° C.) withstirring. The precipitate was collected and washed twice with petroleumether. At this point the crude product contains three components; the2′,3′ disilylated, 2′ silylated (major product) and 3′ silylated. Thepure 2′ protected isomer were obtained by silica gel columnchromatography. This product is then ready for phosphitylation.

TheN-protected-5′-O-dimethoxytrityl-2′-O-tert-butyldimethylsilyl-ribonucleosideswas dried by two co-evaporations with anhydrous pyridine and THF. Theresidue was dissolved in anhydrous THF under argon.Dimethylaminopyridine, N,N,N-ethyldiisopropylamine andcyano-ethoxydiisopropy amino-chlorophosphine were added through a rubberseptum. After 2 hours the reaction mixture was quenched with ethylacetate and washed with 5% sodium bicarbonate followed by water. Aqueouswashes were back extracted with ethyl acetate. Combined organic layerswere dried over sodium sulphate. Solvent was evaporated yielding aviscous oil. The product was co-evaporated twice with toluene and thepale yellow phosphoramidite products were purified by flash silica gelchromatography.

Generalized Sugar Protection and Phosphinylation of Modified Nucleotides

The protecting group is subsequently removed after synthesis of the RNAoligomer. The addition of a protecting group to each modified base andribose is described. While 2 position thio-groups can be oxidized instandard RNA synthesis protocols this has been overcome by using thetert-butyl hydroperoxide (10% solution in acetonitrile) oxidizing agent(Kumar and Davis, 1997).

The modified bases were incorporated into the 17 residue tRNA oligomersbased on 7 food and water borne bacteria from the Category B prioritypathogen list (Table 2, S=mnm5s2U; 6=t6A; $=cmnm5s2U; P=pseudouridine)along with a random 17mer oligomer to be used as a negative control. Inthose cases where the actual oligomer sequence was not fully determined(S or $ in Table 3) both sequences were synthesized and tested. Eacholigomer was tagged with a fluorescent dye for use in the assays anddiagnostic. In addition, a subset of the oligomers were synthesized witha thiol group on the 3′ end and a different dye for attachment to themicroarray chips. For the microtiter plate format each oligomer wastagged with fluorescent dye on the 3′ end. Purity of the oligomers wasconfirmed by gel electrophoresis and proper incorporation of themodified nucleotide bases was confirmed by mass spectrometry.

TABLE 3 (SEQ ID NOS 9-17), respectively in order of appearance): E. ColiG U U G A C U S    U U 6 A P C A A U Salmonella G U U G A C U S    U U 6A P C A A U Shigella G U U G A C U S    U U 6 A P C A A U CamptobacteriaU C U C C C U Sor$ U U 6 A G G A G G Vibrio G U U G G C U Sor$ U U 6 A CC A A U Listeria 1 U C U G A C U Sor$ U U 6 A P C A G A Listeria 2 G C UG A C U C    U U 6 A P C A G C Yersinia 1 C U U G A C U S    U U 6 A P CA A U Yersinia 2 C U U G A C U C    U U 6 A P C A A U

Example 2 Inhibitor Screening Assay

A set of experiments was conducted to identify an RNA oligomer substrateto be used in an assay to identify substrates for use in identifyinginhibitors of tRNA binding to a phenylalanine synthetase from yeast. Apeptide was first identified for use as a phenylalanine synthetasemimic. The peptide was labeled with a fluorescent peptide for use indetection in binding assays.

A series of RNA oligomer substrates were also synthesized containingmodified nucleotides (FIG. 2). A 17mer RNA oligomer containing themodified nucleotide bases Cm, Gm, and m5C was identified as having agreater affinity for a fluorescent peptide that mimics phenylalaninesynthetase than the native RNA oligomer or the unmodified RNA oligomerwith no modified nucleotide bases (FIG. 2).

To determine binding affinity, an assay of ASL for yeast tRNA^(phe) wasprepared varying only in the number of post-transcriptional nucleotidebase modifications. This array was probed using a peptide from a phagedisplay library that bound to an ASL containing 3 modifications. Theaffect of modification on the affinity of a peptide to an RNAsubstrate-sequence (Sequence A in FIG. 2: 5′-CCAGACUGAAGAUCUGG) (SEQ IDNO: 18) is the primary unmodified yeast ASL^(Phe). Sequence B in FIG. 2(5′-CCAGACmUGm AAGAUm⁵CUGG) (SEQ ID NO: 19) is a template ASL sequenceindicating the locations of the modified nucleotides for the variouscombinations of modified nucleotide bases in the mimics of the anticodonstem loop. Sequence C in FIG. 2 (5′-CCAGACmUGmAAm1GAΨm⁵CUGG) (SEQ ID NO:20) is the sequence of the native yeast tRNA^(phe). Sequence D in FIG. 2(5′-GGUCUAGAA GmUCmAGACC) (SEQ ID NO: 21) is a secondary structure forthe doubly modified Phe ASL sequence that's has a propensity to form aduplex. When the affinity of the peptide to the ASLs was determined itwas found that the modifications affected the affinity by more than 3orders of magnitude (FIG. 2). This assay also demonstrates thesignificance of the effect of the modified nucleotides on bindingbetween the enzyme and its RNA substrate. Such an assay may be used toidentify inhibitors of tRNA^(phe) binding to phenylalanine synthetase.

Example 3 Lysine Synthetase Inhibitor Assay

A set of assays can be developed to target LysRS that is unique to eachof the Category B Bacteria and can be based on microtiter platetechnology. In addition, assays can be developed to utilize microarraytechnology for the development of a multidimensional HTS assay thatincorporates a subset of 7 of the individual HTS assays onto a singlemicroarray chip. A diagnostic assay can also use the microarraytechnology, but in a format tailored to diagnostic applications. Themicrotiter based and microarray based HTS assays can then be used toscreen compound libraries for active compounds that can be developed asa narrow-spectrum organism specific antibiotics and as a broad-spectrumantibiotic.

RNA oligomers are produced incorporating modified nucleotide bases,mnm⁵s²U, cmnm⁵s^(2U), t^(6A), and P. The modified bases are incorporatedinto the 17 residue tRNA oligomers in Table 3 along with a random 17meroligomer to be used as a negative control. In those cases where theactual oligomer sequence has not been fully determined (S or $ in Table3) both sequences are synthesized and tested. Each oligomer is taggedwith a fluorescent dye for use in the assays and diagnostic. Inaddition, a subset of the oligomers is synthesized with a thiol group onthe 3′ end and a different dye for attachment to the microarray chips.For the microtiter plate format each oligomer is tagged with fluorescentdye on the 3′ end. The RNA oligomers are synthesized and purifiedfollowing protocols developed specifically for these modified reagents(Agris et. al., Biochimie, 77(1-2):125-134 (1995), Murphy et. al., Nat.Struct. Mol. Biol., (12):1186-1191 (2004)). Purification of theoligomers is performed by HPLC as described (Agris et. al., Acta BiochimPol., 46(1):163-172 (1999)). Purity of the oligomers is confirmed by gelelectrophoresis and proper incorporation of the modified nucleotidebases is confirmed by mass spectrometry.

LysRS corresponding to each of the 7 organisms will be over expressed inand isolated from E. coli using previously described methods (Madore et.al., FEBS Journal, 266:1125-1135 (1999)). A BAC clone containing theLysRS gene can be obtained. The LysRS is encoded from nucleotides fromeach of the organisms and is available on a BAC clone (Table). From theBAC a subclone can be used to generate an expression vector forproduction of the Lysine Synthetase enzyme. The production of activeLysRS enzyme can be performed using published protocols (e.g. Madore et.al.).

TABLE 4 LysRS genome for each organism is available as a BAC clone.Organism Genome location Source Salmonella enterica 19802-21330Washington University Genome Sequencing Center. St. Louis, MoCampylobacter jejuni 365440-367864 Sanger Inst. Hinxton, Cambridge, UKListeria 35510-37065 Institut Pasteur Paris, monocytogenes chromosome I,France Vibrio cholerae section 239 The Institute for Genomic residues7399- Research Rockville, 10352 Maryland Escherichia coli3838117-3841613 Laboratory of Genetics, O157: H7 IP32953 University ofWisconsin- Madison, Madison Wisconsin. Yersinia 3718692-3722057 InstitutPasteur Paris, pseudotuberculosis France Shigella flexneri 2a str.2958664-962160 Laboratory of Genetics, 2457T University of Wisconsin-Madison, Madison Wisconsin

Assays can then be performed to monitor the inhibition of ASL^(Lys)binding to LysRS. Such assays take advantage of the fact that modifiedtRNA^(Lys) is a 100 fold better substrate for binding to LysRS than atRNA^(Lys) containing no modified bases (Sylver et. al., Biochemistry32(15):3836-3841 (1995)). Monitoring of the ASL^(Lys) LysRS complexformation can be determined using time-resolved fluorescent detection(Millar, Curr. Opin. Struct. Biol., 6:637-642 (1996)). Time-resolvedfluorescence detection allows for monitoring complex formation andinhibition in solution. Affinity of each of three or four oligomers foreach LysRS: modified ASL^(Lys); unmodified ASL^(Lys); and, the negativerandom control can be determined by monitoring the change influorescence.

The enzyme and tRNA interactions can be monitored using time resolvedfluorescence spectroscopy to effectively monitor the binding of tRNA tothe synthetase (Lam, Biochemistry, 14:2775-2780 (1975)). tRNA synthetasebinding can be characterized by monitoring the quenching of tryptophanresidues in the protein. While several of the target organismsynthetases contain a tryptophan that could be used to monitor tRNAbinding, for uniformity the LysRSs is labeled using fluoresceinisothiocyanate, FITC (Pierce, Rockford Ill.), as described, for example,by Commans, J. Mol. Biol., 253:100-113 (1995). The association of tRNAfor its cognate synthetase is strong (Ka=1×10⁶) and specific withnon-cognate tRNA Ka 1 to four orders of magnitude lower. While measuringtotal fluorescence is sufficient to monitor complex formation,integration of time resolved fluorescence detection allows formonitoring both free and bound species in the interaction (Jager, Curr.Pharma. Biotech., 4:463-476 (2003)). Time resolved measurements monitoran intrinsic molecular property which results in high statisticalaccuracy and may make fluorescence lifetime analysis (FLA) moreappropriate for HTS applications (Jager). Titrations studies 100 nM ofenzyme solution can be used to monitor complex formation (Commans 1995).To determine the amount of ASL to add per reaction a titration of theASL can be made. The starting solution conditions for the assay can be 5mM MgCl₂ and 2 μM spermidine, pH 6.8. Affinity of tRNALys for thesynthetase a titration of 0 to 4 μM should be sufficient to achievecomplete complex formation. Initially a 96 well format can be used inmethod development that will be expanded to a 386 format once the assayparameters have been determined.

Two alternatives are available for time resolved fluorescence. First, isthe use of alternative fluorescent dyes such as Cy3 or Cy5 which caneasily be attached to each oligomer. Alternatively, an enzyme linkedassay format is available where the purified LysRS can be chemicallymodified to add biotin which will bind to the surface of streptavidincoated well using a Mts-Atf-Biotin Label Transfer Reagent from Pierce,Rockford Ill. This will allow for the binding of the protein to thesurface of each well. With the LysRS bound to surface, complex formationcan be monitored.

Using the more sensitive assay protocol from above, the assay reagentsand conditions can be varied and the optimum reaction mixture will bedetermined based on Z-factor analysis. After the optimum conditions aredetermined, the robustness of the assay will be verified by modifyingvarious reagent concentrations or assay conditions by .+−0.10% anddetermining Z-factor scores. These scores will be plotted and used todetermine which assay conditions are most relevant to control. Inparallel, positive control compounds can be tested and selected. Thepositive control compounds can be used during the routine use of theassay to verify that the assay is functioning properly. As part of theassay optimization experiments, the specificity and selectivity of eachenzyme can be confirmed by determining the affinity for other closelyrelated substrates (oligomers prepared for the other LysRS).

The assays can then be used with a small number of molecular inhibitorscontained in a test library supplemented with known inhibitors of LysRSalong with other known positive controls to confirm the use of theassay. The test compounds, known AaRS inhibitors and positive controlcompounds can be aliquoted into 96-well plates at a predeterminedconcentration corresponding to 40 μg of compound per well. The remainingassay components can be added and the reaction allowed to proceed tocompletion. The reaction results can be quantified and the IC₅₀calculated. A dilution series of compounds with an IC₅₀ less than 1×10⁻⁵M can be analyzed with this assay to confirm that the compound is activeand to more accurately determine the IC₅₀. Those compounds that areactive in this assay can be classified as ‘hits’ and further assayed forbiological testing to determine antimicrobial activity.

For determining activity of the compounds, the following bacterialspecies can be tested against each sample:

Escherichia coli American Type Culture Collection (ATCC) 25922 as asurrogate marker for diarrheagenic E. coli.

Shigella sonnei (clinical isolate) as the most common species causingshigellosis in developed countries.

Salmonella serotype Enteritidis (clinical isolate) as a common andexpanding global cause of salmonellosis.

Yersinia enterocolitica (clinical isolate) as a common cause ofintestinal yersiniosis.

Vibro cholerae (clinical isolate) as the most important species in thegenus Vibrio causing diarrhea.

Campylobacter jejuni (clinical isolate) which continues to be the mostcommon enteric pathogen isolated from patients with diarrhea.

Listeria monocytogenes (clinical isolate) as the most common cause ofinvasive listeriosis.

As a minimum inhibitory concentration (MIC), an achievable potency ofless than or equal to 32 μg/ml tested against any of the above pathogenswould be considered an active compound worthy of an extended secondaryscreen study.

Samples can be tested using the reference broth microdilution methodsrecommended by the Clinical and Laboratory Standards Institute (CLSI;formerly the National Committee for Clinical Laboratory Standards[NCCLS]) M7-A7 and M45-A documents. A 96 well microtiter tray assay canbe used to test a single concentration of 32 μg/ml against eachpathogen. A working concentration of 64 μg/ml can be made usingappropriate solvents and diluents. A calibrated pipette is used totransfer 50 μL of each sample into one well of each of three 96-wellmicrotitre plates. A direct colony suspension is used to make a standardinoculum (equivalent to a 0.5 MacFarland standard) of each bacteria inMueller-Hinton broth (MHB). A 50 μL aliquot of a diluted bacterialsuspension in MHB (supplemented with lysed horse blood for C. jejuni) isadded to each sample to achieve a final bacterial concentration of3-5×10⁵ CFU/ml, thus diluting the sample 1:2 to a final testconcentration of 32 μg/ml. Each batch of samples includes two internalquality control antimicrobial agents with a know potency range and thattarget protein synthesis. A positive growth control with only growthsupport media and an ethanol control at concentrations equivalent tothat in the samples is also tested for each pathogen concurrently. Afterthe broth microdilution plates are inoculated and incubated in anambient air environment at 35° C. for 20-24 hours for the entericbacilli and L. monocytogenes. C. jejuni is incubated for 48 hours at 37°C. in a microaerobic atmosphere, using gas-generated sachets in a sealedhard plastic container. After appropriate incubation times, the platesare removed from incubation and each well inspected for growth. If awell is clear of growth (non-turbid), an MIC of .ltoreq.32 μg/ml wouldbe determined and the investigational agent therefore identified asbeing active

A secondary screen of “active” samples includes an extended dilutionseries (eight to 12 log₂ dilution steps) to determine “on-scale” MICvalues for a potential antimicrobial agent including evaluation ofbreadth of spectrum against a larger collection (140 strains) of FWBpathogens listed above (20 isolates each). These isolates are recentclinical strains representing wild-type and strains with documentedcritical resistance phenotypes; testing methodologies to be thosedescribed above.

A multidimensional microarray HTS based on the above assay can then bedeveloped. The multidimensional microarray HTS can be used to screen forinhibitors of each enzyme simultaneously; thus, providing a means toidentify narrow spectrum inhibitors (inhibits one enzyme) or broadspectrum inhibitors (inhibits several to all of the enzymes). Each ofthe specific oligomers can be attached in a small grid on themicroarray. After all of the oligomers are attached each enzyme is addedand allowed to bind to its respective oligomer to form asubstrate-enzyme complex as confirmed in the specificity and selectivitytesting. A single inhibitor is then added to each grid and the activecompounds will dissociate the substrate-enzyme complex as determined bymeasuring fluorescence.

Alternatively, rather than fluorescently labeling the bacterialproteins, a fluorophore can be attached within the RNA substrate. Whilethe most common method to fluorescently label an oligomer is to attachit either on the 3′ or 5′ end; for these microarray applicationsattaching the fluorophore within the oligonucleotide may simplify and/orfacilitate detection. A pyrrolo fluorescent cytidine analog may be usedin the generation of the RNA oligonucleotides (Glen Research Sterling,Va.). This cytidine analog base pairs with other nucleotides as a normalnucleotide. Also, this fully substituted oligonucleotide has the same Tmas the control oligonucleotide.

A review of all the LysRS substrates, Table 3, finds that they allcontain a cytosine in the loop at position 32 and also in the stemportion of the ASL. To determine to optimum position for placement ofthe cytidine analog, test oligomers can be made with both positionslabeled for the E. coli substrate. The association constant andintensity of signal quenching can be determined for both test oligomers.Results of these experiments will determine the optimal substrate(s) forthe chip. For the remaining oligomers, the selectivity and specificityof each oligomer containing the modified nucleotide bases to theirrespective enzymes is confirmed in solution during the development ofthe HTS assays and can be verified when attached to the microarray chip.The organism specific RNA oligomers are attached to the glass microarraychip using gold-thiol methods. The thiol group is commercially availablein the phosphoramidite formulation used in oligomer synthesis and isincorporated in the same chemical step fashion as individual amidites(i.e. A, U, G, C) during synthesis of the oligomers.

Each individual oligomer along with a positive and negative control willbe attached to a substrate followed by addition of the enzymes. Eachgrid is mechanically isolated. Inhibitors are added to each gridfollowed by fluorescent detection to determine which enzyme-substratecombinations are dissociated; thus, identifying active inhibitors.

A rapid diagnostic assay can also be developed using the same microarrayand the organism specific RNA oligomers used for the HTS screeningassays. To use the assay, a subsample of the specimen extract is appliedto the microarray after which the microarray is rinsed and analyzed withfluorescence detection. If a particular spot fluoresces then the sampleis positive for the organism corresponding to that spot. The arrangementof the oligomer substrates on the microarray can be arranged dependingon the particular application of the diagnostic assay.

Culture media containing each organism is extracted to release theproteins from the organisms. A subsample of this extract is applied toeach grid on the microarray containing RNA oligomers. The microarray isrinsed with distilled water and analyzed with a standard laboratoryfluorometer. Those spots corresponding to the specific organism shouldfluoresce. This process can be repeated for each organism to confirmselectivity and specificity. Finally, sensitivity testing can beconducted by repeating these assays using cultures with decreasingnumbers of plaque forming units and/or extract dilutions.

1. A microarray comprising a plurality of nucleic acid molecules havingat least one modified nucleotide and a solid support to which theplurality of nucleic acid molecules are attached, wherein the nucleicacid molecules are tRNA fragments having between 5 and 25 continuousnucleotides of tRNA molecules, and wherein the nucleic acid moleculescomprise fragments of tRNA molecules consisting essentially ofTΨC-loops, D-loops, and anticodon stem loops.
 2. The microarray of claim1, wherein the nucleic acid molecules are tRNA fragments having between5 and 25 continuous nucleotides of tRNA^(Ala), tRNA^(Arg), tRNA^(Asn),tRNA^(Asp), tRNA^(Cys), tRNA^(Gln), tRNA^(Glu), tRNA^(Gly), tRNA^(His),tRNA^(Ile), tRNA^(Leu), tRNA^(Lys), tRNA^(Met), tRNA^(Phe), tRNA^(Pro),tRNA^(ser), tRNA^(Thr), tRNA^(Trp), tRNA^(Tyr), and tRNA^(Val).
 3. Themicroarray of claim 1, wherein the nucleic acid molecules include adetectable label.
 4. An isolated nucleic acid molecule comprising anucleic acid fragment having between 5 and 25 continuous nucleotides ofa tRNA D-loop having at least one modified nucleotide.
 5. The isolatednucleic acid molecule of claim 4, wherein said nucleic acid fragment isa fragment of 16 nucleotides.
 6. The isolated nucleic acid molecule ofclaim 4, wherein the at least one modified nucleotide is dihydrouridine.7. The isolated nucleic acid molecule of claim 4, wherein the nucleicacid fragment is capable of forming a stem-loop structure.
 8. Theisolated nucleic acid molecule of claim 4, wherein the nucleic acidmolecule comprises a nucleic acid molecule having the sequence 5′-m′n′o′p′NN(dihydrouridine) NN(dihydrouridine)NNp″o″n″m″, wherein m′, n′, o′,p′ refer to any nucleotide and are complementary to m″, n″ o″ and p″,and N refers to any nucleotide.
 9. An isolated nucleic acid moleculecomprising a nucleic acid fragment having between 5 and 25 continuousnucleotides of a tRNA TΨC-loop having at least one modified nucleotide.10. The isolated nucleic acid molecule of claim 9, wherein said nucleicacid fragment is a fragment of 17 nucleotides.
 11. The isolated nucleicacid molecule of claim 9, wherein the nucleic acid fragment has at leasttwo different modified nucleotides.
 12. The isolated nucleic acidmolecule of claim 9, wherein the at least one modified nucleotide isselected from the group consisting of Psi, ribothymidine, and m1A. 13.The isolated nucleic acid molecule of claim 9, wherein the nucleic acidfragment is capable of forming a stem-loop structure.
 14. The isolatednucleic acid molecule of claim 13, wherein the nucleic acid moleculecomprises a nucleic acid molecule having the sequence 5′-h′IT k′l′(ribothymidine)(Psi) CN(m1A)NNl″k″j″I″h″, wherein h′, i′, j′, k′, and l′refer to any nucleotide and are complementary to h″, i″, j″, k″, and l″,and N refers to any nucleotide.